DE2723165B2 - Process for the preparation of a polysaccharide - Google Patents

Description

40

The invention relates to a method for producing an alginate-like polysaccharide by cultivation of bacteria of the species Azobacter vinelandii.

Alginic acid, a hydrophilic colloidal hydrocarbon acid, consists of a different block copolymer composed of D-mannuronic acid units and L-guluronic acid units. Although alginic acid itself is virtually insoluble in water, it can can be made easily soluble by neutralization with a suitable alkali. One of the special properties of alginate solutions is their high viscosity at very low concentrations; with the addition of certain divalent ions, such as calcium or Magnesium to solutions of alginates, a gelation is initiated. The unusual physical Properties of alginic acid and alginates allow a wide range of technical applications as Emulsifiers, stabilizers and thickeners. You can for example in the food industry be used as emulsion stabilizers for ice cream, as gelling agents for milk pudding and as Thickeners for sauces and as foam stabilizers for beer; pharmaceutically they can be used as Emulsifiers and granulating agents for tablets, as suspending agents for ointments and as absorbable Gels can be used for surgical dressings. In paper and textile processing they are called Glues, for coating compounds, coating compounds, Dyes and printing inks are used and in agriculture they can be used as soil improvers.

Alginates and alginic acids are technically made by extracting certain species of brown seaweed, for example Laminaria digitata and Ascophyllum nodosum obtained, whereby they a considerable proportion of the cell walls.

This common extraction method of seaweed has the disadvantage that one on the supply of Alginate-containing seaweed is dependent as the starting material. The process can only be used difficult to carry out because of the amounts of seaweed being processed and a considerable additional Cleaning is sometimes required, especially when a material is required for food and pharmaceutical applications.

Because of these difficulties, alginate-like polysaccharides have been produced in recent years been obtained by cultivating various species of Azotobacter, particularly Azotobacter vinelandii. The alginic acid produced by these microorganisms is structurally similar to that of seaweed obtained, with the exception that it is partially acetylated

It is known that the production of polysaccharides using a strain of Azotobacter vinelandii is critically dependent on a number of factors, in particular the pH of the medium and also on the amount of phosphate in the medium.

In GB-PS 13 31 771 a process for the production of polysaccharides is described which consists of a partially acetylated variable block copolymer of 1-4-linked D-mannuronic acid and L-guiuronic acid reFten, there is one microorganism of the species Azotobacter vinelandii under aerobic conditions in an aqueous nutrient medium containing at least one monosaccharide and / or disaccharide as a carbon source and containing as essential Ingredients Sources of molybdenum, iron, phosphate, magnesium, potassium, sodium, calcium and sulfate, among controlled pH conditions so that a pH in the range 6.5 to 8.5 for at least the first half the fermentation period and in the range from 4.5 to 8.5 for the remainder of the fermentation period, if one is present, cultivation is carried out until a substantial one Formation of the polysaccharide has taken place, whereupon the formed polysaccharide is isolated. The one in this The method of cultivation used may be batch or continuous.

In the description it is emphasized that the amount of Phosphate in the growth medium plays an essential role in terms of the yield of polysaccharide It is suggested in the description there that low amounts of phosphate increase the yield and it becomes a phosphate concentration of 0.015 to 1.4 mmoles, preferably 0.2 to 0.7 mmoles, are suggested. Polysaccharide concentrations of about 2.5 to 3.0 g / l are obtained under these conditions.

In GB-PS 13 34 413 a special selection of the conditions to achieve particularly good Described yields of polysaccharides. In this patent, a method similar to that in described and claimed in the aforementioned patent, but the concentration of phosphate in the Medium is limited to 0.1-0.8 mmol and during of cultivation, the pH of the medium is in the range

from 7.0 to 8.2. Under these conditions pplysaccharide concentrations of 3 to 3.5 g / l can be achieved can be obtained.

In GB-RS 1394413 it is again emphasized that the best results are obtained at low phosphate concentrations.

It has now been found that in a continuous separation process of this type, the actual concentration of phosphate in the medium falls below certain conditions can be considerably higher than was emphasized in the previous patent specifications.

Under continuous release conditions it is an essential requirement that the culture be carried out under limited phosphate conditions. This means that it is the amount of phosphate in the medium which is the determining factor for the Concentration of the microorganism becomes.

It should be noted that the concentration on Cells of the microorganism present in the ferment function as a function of the concentration of the phosphate is what boundary conditions result in a continuous culture The higher the phosphate concentration, the higher the concentration of cells present under boundary conditions. Provided this principle is applied, it is possible to create phosphate-limited conditions in continuous crops, with phosphate quantities, which are significantly in excess of those disclosed in the prior patents and where much higher polysaccharide concentrations, for example up to 27 g / l polysaccharides, are achieved, especially if lower degrees of dilution are used

According to the present invention, a method for producing a polysaccharide from a partially acetylated variable block copolymers 1-4 linked D-mannuron and L-guluronic acid residues provided in the one bacterium of the species Azotobacter vinelandii under aerobic conditions in an aqueous culture medium containing at least one essential component Monosaccharide or a disaccharide as a carbon source and sources of phosphate molybdenum, iron, Magnesium, potassium, sodium, calcium, and sulfate, are continuously cultivated, with the aqueous Culture medium contains a certain amount of nitrogen and / or nitrogen-containing aeration gas and the concentration of phosphate in the medium has a limiting effect on the concentration of bacteria and is at least 1.0 mMol, and wherein during cultivation the pH of the medium is in the range of 6.0 to 8.2 is held.

Any strain of Azotobacter vinelandii can be used in the method of the invention. Particularly valuable trunks, soft particularly good ones Yields of the polysaccharide are those mentioned in the two earlier patents, which the culture collector's no. NCIB 9068 and NCIB 8799 carry, as well as the strain, which the culture collection no. NCIB 8660 carries These strains are available from the National Collection of Industrial Bacteria, Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen, Scotland, and are described in the collections catalog.

The amount of phosphate used in the medium varies depending on the cell concentration in the fermentation vessel: the higher the Cell concentration, the higher the amount of phosphate, which is limiting It has been found, for example, that when using a chemically defined medium of the type as described in Example 1 of GB-PS 13 31771, a phosphate concentration of 1.0 mmol and a dilution rate A speed of 0.15 l / h is effectively limiting at a cell concentration of around 23 g / L

A phosphate concentration of 2.0 mmol is effective limiting at a cell concentration of about 4 g / l, while a phosphate concentration that is so high As 2, OmMoI is effective limiting at a cell concentration of about 4.7 g / l is at lower At the same degree of dilution, the cell concentration increases, i.e. the total cell mass in a specific

is limiting phosphate concentration, probably by increasing the individual cell weight. Thus, at a degree of dilution of 0.05 l / h 3.0 mM phosphate limiting at a cell concentration of about 14 g / l.

In any particular case, there can be an indication of whether the phosphate concentration is actually limiting by increasing the phosphate concentration and observing the effect on the cell concentration be recognized. When the concentration isn't one limiting component in the medium is increased, no significant change in cell concentration is observed; however, the concentration of limiting component increases so the cell concentration responds very quickly through an increase.

For example, it causes an increase in the limiting Phosphate concentration around 2 times at least one

10% increase in cell concentration within the 5th Hours.

It has been found that under conventional continuous

Chen cultivation conditions, for example using a chemostat (see Herbert EIsworth and Teilung, 1956, Journal of General Microbiology, 14,601) particularly advantageous amounts of phosphate are in the range from 2.0 to 3.0 mmol, with a cell concentration of about 4 to about 4.7 g / l at dilution levels of 0.15 h- 'or about 14 g / l Achieved at 0.05 hr. Using these amounts, a folysaccharide concentration of up to about 27 g / l found. There can be no upper limit of the Phosphate concentration should be determined, taken from the limit dictated by practical considerations as essential in continuous fermentation processes. In the aforementioned earlier patent specification

so indicated that another factor is important in the formation of polysaccharide using Azotobacter vinelandii, namely the amount of oxygen added to the fermentation system will. Oxygen solution amounts of about 5 to 50, preferably about 8 to about 20 millimoles of oxygen per liter Culture medium per hour are mentioned word for word

It has been found that the effectiveness with which the monosaccharide or disaccharide used in a polysaccharide is converted, is related to the amount of oxygen supply. With continuous Cultivation conditions are the same for oxygen, as is also true for phosphate, namely that one There is a relationship between the oxygen content and the cell concentration which is essential and not the oxygen concentration in the medium as such. It was found that although an oxygen supply of about 50 mmol / g cell / h under phosphate-limiting conditions a sucrose conversion of about

9% results, but that an oxygen supply of about 7 to about 22 mmol / g cell / h a sucrose conversion of over 40% results in an oxygen supply of 20.5 mmol / g cell / h has a sucrose conversion of about 49%.

The actual oxygen uptake in mmol / l / h according to these numbers, of course, depends on the cell concentration in the fermentation vessel. The intake per l / h is generally in the previous patents described area, but it must be emphasized that there is the relationship between Oxygen delivery and cell concentration is that which is essential

Altering the oxygen supply to the culture can be effected in a number of ways. First can reduce the concentration of oxygen in the gas, generally air, which entered the culture during the Fermentation is initiated, can be changed by blowing the air with nitrogen or an inert gas diluted or by enriching the air with extra oxygen. Furthermore, the pressure of the in Change the culture of the introduced gas so that the rate of dissolution varies. Thirdly, the Effectiveness with which the oxygen in the gas phase is converted into the liquid phase is changed by changing the gas-liquid mixing properties, which is most easily done by using a Changing the speed and effectiveness of the stirrer is a combination of two or more of these procedures can also be applied to find the correct balance between Ensure nitrogen and oxygen.

To get the polysaccharide in high quantities and good effectiveness in terms of conversion

Table 1

Composition of the growth medium

To maintain the mono- or disaccharide is it required, the growth rate of the bacteria to monitor and separately the degree of respiration, so that the supply of oxygen to the Bacteria on the one hand is not limited and on the other hand is not unnecessarily high. It must be emphasized that the oxygen supply does not have to be limiting because it lowers the formation of the polysaccharide. On the other hand, unnecessarily high degrees of respiration, which result from a high oxygen content, that a large proportion of the carbohydrate source is oxidized to carbon dioxide, whereby a low Conversion effectiveness results

The invention is explained in more detail in the following examples

Examples 1 to 4

Different media according to Table 1 were used:

The media were prepared and added to the cultures in two batches: batch (1) was autoclaved at 1 kg / cm ² for 1 hour in two parts, which were aseptically combined after cooling. One part contained sucrose, KH ₂ PO ₄ and K ₂ HPO ₄ in 141, the other part contained MgSO ₄ · NaCl and trace elements with the exception of calcium and iron in 4 L batch (2) contained CaCl ₂ and FeCl ₂ in 21; the CaCl ₂ was autoclaved in 1.91 and the FeCl ₂ was filter-sterilized in 100 m! and then added to the mass of the approach (2). Batches (1) and (2) were added to the culture medium through separate lines, with (1) being added at 9 times the rate of (2) so that the concentration in the culture as shown in Table 1 was established.

Ingredient concentration (g / l) [millimolar] Example 1 Example 2 Example 3 Example 4 Example p

Sucrose

KH ₂ PO ₄

K ₂ HPO ₄

MgSO ₄ 7H ₂ O

Na ₂ MoO ₄

CaCl ₂ 2H ₂ O

FeCl ₂ 4H ₂ O

H ₃ BO ₄

CoSO ₄ 7H ₂ O

MnCl ₂ 4H ₂ O

ZnSO ₄ 7H ₂ O

CuSO ₄ 7H ₂ O

FeSO ₄ 7H ₂ O

Using four continuous chemostat-type cultures (Herbert et al, as previously mentioned) of Azotobacter vinelandii NCIB 9068 in a continuous culture device with one culture volume of 1.01 were each mixed with one of the sterile media as indicated in Table 1, namely with the phosphate concentrations given there.

60 60 60 120 20th 0.0321
0.12 y ^ ⁰⁶⁴ } [2.0]
0.25 y ° ' ^O96 1 [3, O]
0.38 y ÖD ^{13 '01} 0.0081
0.032 J. 1.6 1.6 1.6 1.6 0.2 1.6 1.6 1.6 1.6 0.2 0.008 0.008 0.008 0.008 0.008 0.34 0.34 0.34 0.34 0.043 0.017 0.017 0.017 0.025 - 0.023 0.023 0.023 0.23 - 0.009 0.009 0.009 0.009 - 0.007 0.007 0.007 0.007 - 0.009 0.009 0.009 0.009 - 0.0008 0.0008 0.0008 0.0008 -

65 0.003

Mixtures (1) and (2) were pumped to the cultures in a combined amount to obtain the degrees of dilution given in Table 2, and the culture broth flowed into a receptacle through a standpipe overflow. The temperature was set as indicated in Table 2
The pH was increased to 7.4 by automatic addition of

1 M NaOh set. Air was bubbled into the fermenter at a rate of 1 liter / min, and the The stirring speed was adjusted so that the acid uptake was in the range from 10 to 30 mmol / g Cell / h. From the fermentation vessel samples were taken daily and investigations of the Cell masses and polysaccharide concentrations. For each 40 ml sample, 0.8 ml Added 0.5 M EDTA and 0.8 ml 5 M NaCl. The samples were then centrifuged at 25,000 g for 40 minutes. In the The resulting cell beads were resuspended in distilled water for 40 minutes at 2,000 g centrifuged and the supernatant was decanted.

Table 2

Polysaccharide formation under phosphate-limited conditions A previously weighed tube containing the sediment was ^{dried for 12 hours at 105 °} C. and then weighed. The polysaccharide was precipitated from the supernatant from the first centrifugation by adding 3 parts by volume of propan-2-ol. The precipitate was collected by filtration and dried in vacuo at 45 ° C for 24 hours. The phosphate limitation was achieved at phosphate concentrations of 1.0 OmMoI to 3.0 mmol. Particularly suitable concentrations of polysaccharides were obtained using media containing 2.0 to 3.0 mmol of added phosphate.

example phosphate Dilution Cultivation Cell concentrations Polysaccharide Polysaccharide concentration Degree temperature tration concentration productivity (p.c.) = d.r. X p.c. (mM) (η " ¹ ) (O (g / l) (g / l) ig / l / h) 1 1 0.15 30th 2.3 4.4 0.66 2 2 0.15 36 4.0 9.1 1.36 3 3 0.12 36 4.7 15.1 1.81 4th 3 0.05 36 14th 26.5 1.32

In all cases it is shown that the phosphate concentration limits the cell concentration by the Phosphate concentration is increased twice: within 5 hours one observed Increase in cell concentration by at least 10%.

Example 5

Effect on the polysaccharide yield by

different ventilation speeds in
Culture vessel under phosphate-limited conditions

Azotobacter vinelandii NCIB 9068 was grown on a continuous culture using an aqueous Culture medium having the composition of Table 1 was grown in a stirred tank fermenter.

A vaccine grown on an agar medium was added to the medium in the fermentation device, and fermentation was carried out in batches for 24 hours at 30 ° C. at a pH of 7.5. A continuous supply of medium was added at a degree of dilution of 0.15h " ¹ (ie 150 ml / h). Air was added to the culture in a

jo amount of 1.7 l / min, given. The degree of respiration the organism was adjusted by changing the stirring speed, whereby the culture one let reach constant stage. Each time a constant stage was reached, the Polysac-

3) charid formation measured by using propan-2-ol a precipitate formed and then determined by dry weight, the cell product became measured by determining the dry weight and the residual sucrose was determined by g. 1. c.

Oxygen uptake was measured using a paramagnetic oxygen analyzer and CO2 evolution was measured using an infrared CO 2 analyzer.
Whenever a constant stage was reached, 2 ml of sterile phosphate solution (0.125M) was added to the culture vessel. In each case the concentration of bacteria increased, indicating that the cell concentration was limited by the phosphate concentration. The results are in

w Table 3 given.

Table 3

Effect of the degree of respiration on the effectiveness of sucrose conversion into a polysaccharide

oxygen Zeil concentration Polysaccharide Used Conversion of Stirrer recording concentration Sucrose Sucrose in speed Polysaccharide (mmol / g cell / h) (g / l) (g / l) (g / l) (Rpm)

7.0
12.4
20.5
28.2
51.0

0.5

0.42

0.44

0.44

0.69

0.70
0.84
1.09
1.16
0.89

1.65

1.8

2.2

4.7

9.9

42%
47%
49%
25%
9%

400
500
600
700
870

From Table 3 it can be seen that the yield of Polysaccharide from sucrose can be increased from 9% at higher degrees of ventilation (and consequently higher degrees of respiration) to 47 to 49% Oxygen uptake between 12 and 20 mmol / g cell / h.

Claims (5)

Patent claims: 1. Process for the preparation of a polysaccharide from a partially ace, ylated variable block copolymer of 1-4 linked D-mannuron and L-guluronic acid residues, in which a bacterium derives from the Species Azotobacter vinelandii is cultivated continuously under aerobic conditions in an aqueous culture medium, the culture medium as essential constituents at least one monosaccharide or disaccharide as a carbon source and Sources of phosphate, molybdenum, iron, magnesium, Contains potassium, sodium, calcium and sulfate, and the medium is a certain source of nitrogen and / or a ventilation gas containing nitrogen, and the pH of the medium is maintained in the range between 6.0 and 8.2, thereby characterized in that the concentration of phosphate in the medium is limiting The concentration of the bacterium is and is at least 1.0 OmMoI 2. The method according to claim 1, characterized in that as A. vinelandii a strain NCIB 9068, 8789 or 8660 is used. 3. The method according to claim 1, characterized in that the phosphate concentration in the Medium is 2.0 to 3.0 mmol 4. The method according to claim 1, characterized in that the oxygen uptake by the Culture is restricted to a level that allows optimal conversion of the monosaccharides or Disaccharide results in the polysaccharide 5. The method according to claim 4, characterized in that the oxygen uptake on a Amount of about 7 to 22 mmol / g cell / h is limited.